Introduction - shodhganga.inflibnet.ac.inshodhganga.inflibnet.ac.in/bitstream/10603/13140/10/10_introduction.pdfAatif M. Ph.D Thesis Introduction 2 Cysteine proteinases (CPs) are the

IInnttrroodduuccttiioonn

Aatif M. Ph.D Thesis Introduction

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1. Introduction 1.1 GENERAL

Proteolytic enzymes, also known as proteinases/peptidases, are enzymes that

catalyze the breakdown of proteins by hydrolysis of peptide bonds. Proteinases are

essential for the survival of all kinds of organisms and are encoded by approximately

2% of all genes [Rawlings et al., 2004]. Proteinases are customarily classified as

exopeptidases when they hydrolyze only the N-terminal or C-terminal bonds in

proteins. They are termed as endopeptidases (or proteinases) when they hydrolyse

internal peptide bonds. The term protease is a general term applied for both

exopeptidase and endopeptidase. A protease is classified as proteinase if it exhibits a

significant degree of endopeptidase activity regardless of whether it exhibits

exopeptidase activity. However, proteinases and proteinases are synonymously used

in literature. Based on the catalytic mechanism, there are four types of proteinase,

serine, cysteine, aspartic and metallo proteinases. Serine proteinases constitute a large

family of enzymes that hydrolyze the peptide bond of substrate via a nucleophilic

serine residue at the active site, Ser-195 (Chymotrypsin numbering), arranged with

Asp-102 and His-57 into a catalytic triad [Hedstorm, 2002]. Serine proteinases have

important role in epidermal homeostasis and skin pathologies [Ovaere et al., 2009].

Cysteine proteinases have a common catalytic mechanism that involves a nucleophilic

cysteine residue in a catalytic triad. Aspartic proteinases essentially use an aspartate

residue for catalysis of their peptide substrates and are known to play important role

in parasite invasion and migration in the host [Yang et al., 2009]. Metallo proteases

are proteolytic enzymes whose catalytic mechanism involves a metal like zinc or

cobalt. A distinct role of matrix metallo proteases has been established in the

development of neuropathic pain [Kawasaki et al., 2008]. The primary role of

proteinases was long considered to be protein degradation relevant to food digestion

and intracellular protein turnover [Barrett et al., 2004]. However, now it is known that

proteinases are involved in the control of large number of key physiological processes

such as cell-cycle progression, cell proliferation and cell death, DNA replication,

tissue remodelling, haemostasis (coagulation), wound healing and immune response

[Turk, 2006].


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Cysteine proteinases (CPs) are the proteins with molecular mass about 21-30

kDa showing the highest hydrolytic activity at pH 4-6.5. CPs are present in all living

organisms. They are synthesized in a precursor form in order to prevent unwanted

proteolysis and later subjected to cotranslational and posttranslational modifications

to convert them into catalytically active mature enzymes [Turk et al., 2000]. Cysteine

proteinases are reported from various organisms which include animals (cathepsins,

calpain), plants (papain, ficin), protozoa (cruzipain), bacteria (streptopain) and viruses

(V-cath proteinase) [Barrett, 2004].

The first clearly recognized and extensively investigated cysteine protease is

papain isolated from the latex of plant Carica papaya.

Mammalian CPs are divided into 4 main groups; namely

1. Lysosomal cathepsins 2. Caspases

3. Calpains 4. Legumain

Cathepsins comprise an important section of the papain family of CPs, sharing

similar amino acid sequences and folds. There are thirteen human cathepsins known

at the sequence level [Turk et al., 2001; Rossi et al., 2004]. Out of which seven viz.,

cathepsins B, H, L, C, O, F and X are ubiquitous i.e. they have a broad tissue

distribution, but they may be involved in more specialized processes [Turk et al.,

2000; Buhling et al., 2000]. Cathepsins are present in the lysosomes of the cells and

are involved in intracellular protein turnover, proteolytic degradation and cleavage of

a number of precursor proteins and hormones [Turk et al., 2000]. Cathepsins K, V and

S are more tissue specific with cathepsin K expressed in osteoclasts only, cathepsin V

in thymus and testis, and cathepsin S in spleen and lung [Turk et al., 2000]. Recently,

cathepsin K was found to be expressed by breast carcinoma cell, mature macrophages,

and multinucleate giant cells adjacent to amyloid deposits in brain [Punturieri et al.,

2000; Rocken et al., 2001].All cathepsins are relatively small monomeric proteins

with molecular mass (MW) in the range of 24-35 kDa, with the exception of cathepsin

C, which is an oligomeric enzyme with MW around 200 kDa [Turk et al., 2002]. All

mature cathepsins are glycosylated at usually one or more glycosylation sites except

cathepsin S. Human cathepsins play very important role in intracellular protein


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turnover in lysosomes and in processing and activation of other proteins including

proteinases, in antigen processing and presentation and in bone remodelling.

However, their specific and individual functions are often associated with their

restricted tissue localization [Brix et al., 2008]. Mutations in cathepsin genes result in

human hereditary diseases such as pycnodystosis (cathepsin K mutation) [Gelb et al.,

1996] and Papillon-Lefevre and Hain-Munk syndromes caused by mutations in the

cathepsin C gene [Allende et al., 2001]. In some pathological conditions like

ischemia, hypervitaminosis and on exposure to UV radiations lysosomal enzymes are

released in extracellular space and produce extensive damage to the extracellular

matrix. There are evidences that blastocyst-derived cathepsins are functionally

involved as zonolytic factors in the hatching of blastocysts in the golden hamster

[Sireesha et al., 2008].

Calpains and Caspases: These are cytoplasmic thiol proteinases. Calpains

participate in many intracellular processes such as turnover of cytoskeletal proteins,

cell differentiation and regulation of signal peptides. They require Ca2+ for activation.

They are ubiquitously distributed and have been implicated in acute neurological

disorders, Alzheimer’s disease, muscular dystrophy and gastric cancer [Huang and

Wang, 2001]. Caspases are cysteine dependent aspartate specific proteinases. They

are involved in cytokine maturation, apoptosis signalling and in apoptosis mediation

[Goyal, 2001].

Legumains: These are a family of asparagine-specific cysteine

endopeptidases involved in pro-polypeptide processing and protein breakdown

[Muntz et al., 2002]. They are involved in MHC class II-restricted antigen

presentation [Manoury et al., 1998] and local negative regulation of osteoclasts

formation and activity [Choi et al., 1994].

1.2 FUNCTIONS OF THIOL PROTEINASES

Lysosomal CPs have been found to be critical for rheumatoid arthritis,

osteoarthritis and osteoporosis [Vasiljeva et al., 2007; Yasuda et al., 2005],

neurological disorders [Nakanishi, 2003], pancreatitis [Van Acker et al., 2002],

cancer [Gocheva and Joyce 2007; Keppler 2006], cardiovascular diseases [Lutgens et

al., 2007].


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In general, cysteine proteinases (CP) are involved in the intracellular

catabolism of peptides and proteins [Barrett and Kirschke 1981], processing of

proenzymes and prohormones [Marks et al., 1986; Taugner et al., 1985], breakdown

of collagen [Etherington, 1980], antigen processing [Turk et al., 2002], muscular

disorders and gingivitis [Turk et al., 1997]. They also have a role in fertilization, cell

proliferation, differentiation and apoptosis [Grzelakowska-Sztaber 1998; Chapman et

al., 1997]. They may play an important role in modulating the penetration and

destruction of tissues by malignant cells during tumor invasion, metastasis [Sloane

and Honn, 1984] and by micro organisms during infection [Barrett et al., 1984].

Proteolytic processing by cysteine proteinases has also been shown to regulate the

assembly of many viral proteins [Korant et al., 1988], including HIV-I [Guy et al.,

1991]. Different functions assigned to cystatin are presented in [Fig. 1].

1.3 REGULATION OF THE ACTIVITY OF THIOL

PROTEINASES

Despite their life-giving functions, the enormous hydrolytic potential of

cathepsins can be damaging in living systems and need to be kept strictly under

control. Failures in biological mechanisms controlling protease activities result in

many diseases such as neurodegeneration, cardiovascular diseases, osteoporosis,

arthritis and cancer. Cells have evolved several distinct mechanisms for the regulation

of excessive CP activity via proper gene transcription, maintenance of the rate of

protease synthesis and degradation and most importantly the interaction of CPs with

the proteins that inhibit them, viz. cysteine proteinase inhibitors or thiol proteinase

inhibitors (CPIs or TPIs) or more commonly cystatins. Cystatin inhibitory activity is

vital for the delicate regulation of normal physiological processes by limiting the

potentially inappropriate activity of their target proteinases, cathepsins, mammalian

legumain and some calpains [Alvarez-Fernandez et al., 1999; Crawford, 1987]. These

proteinase inhibitors are ubiquitously present in the living system and constitute a

powerful regulatory system for the endogenous proteinases. Natural inhibitors of

cysteine proteinases include the members of cystatin superfamily. The cystatin

superfamily comprises a large group of the cystatin domain containing proteins,

present in a wide variety of organisms including humans.


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Figure 1: Diagrammatic representation of the functions of thiol

proteinase inhibitors (cystatins)

The figure shows the role played by cystatins in various

immunological and cellular processes.


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1.4 CYSTEINE PROTEINASE INHIBITORS: THE CYSTATIN

SUPERFAMILY

The name “cystatin” was firstly proposed by Alan J. Barrett [1981] to describe

a small protein isolated from chicken egg-white and demonstrating the ability to

inhibit the activity of lysosomal cysteine proteinases [Barrett, 1981]. Later, proteins of

low-MW and high-MW, capable of inhibiting the activity of cysteine proteinases and

evolved from a cognate ancestral gene, were grouped in a new superfamily of proteins

called the cystatin superfamily. Other cysteine proteinase inhibitors were reported

independently in several laboratories during the period of 70-80’s [Lenney et al.,

1979; Sasaki et al., 1977; Fraki, 1976].

1.5 DISCOVERY OF THE CYSTATIN SUPERFAMILY

Hayashi et al. [1960] for the first time reported the presence of a factor

capable of inhibiting the clotting activity of thiol proteases in mammalian system. The

first isolated and partially characterized protein inhibitor of CPs was from chicken egg

white and was shown to inhibit papain, ficin [Fossum and Whitaker, 1968; Sen and

Whitaker, 1973] and cathepsins B and C [Keilova and Tomasek, 1975]. Later for the

same protein term cystatin was proposed because of its unique property of arresting

the activity of CPs [Barrett, 1981].

The first intracellular protein inhibitor of papain, cathepsin B and H was

isolated and partially characterized from pig leucocytes and spleen [Kopitar et al.,

1978]. The determined amino acid sequences of chicken cystatin [Turk et al., 1983;

Schwabe et al., 1984] and human stefin (stefin A) from the cytosol of

polymorphonuclear granulocytes [Machleidt et al., 1983] confirmed structural

differences between these two homologous proteins. At the same time, inhibitors of

CPs were isolated from sera of patients suffering from autoimmune diseases [Turk et

al., 1983] and based on its sequence homology with chicken cystatin the name human

cystatin was proposed [Brzin et al., 1984], soon renamed to human cystatin C (HCC)

[Barrett et al., 1984]. Also, sequences of bovine and human kininogens were

determined [Nawa et al., 1983] and the concept of cystatin “superfamily”, precipitated

by an observation that multiple cystatin-like sequences were present in kininogens


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and that the stefins were related to both the cystatins and the repeats of kininogens

[Ohkubo et al., 1984]. This data and First International Symposium on Cysteine

Proteinases and their Inhibitors [Portoroz, Yugoslavia- now Slovenia, September

1985, organized by V. Turk] were crucial for the nomenclature and classification of

the cystatin superfamily [Barrett et al., 1986].

1.6 CLASSIFICATION OF THE CYSTATIN SUPERFAMILY

The first classification of the cystatin superfamily into three families was

based on at least 50% sequence identity, inhibition of their target enzymes and

presence or absence of disulphide bonds [Barrett et al., 1986]. Three distinct families

of the protein inhibitors comprise: family 1 or the stefin family, family 2 or the

cystatin family and family 3 or the kininogen family [Fig. 2]. Studies have revealed

the presence of a single domain structure responsible for the inhibitory action in case

of family 1 and family 2 (stefins and cystatin). However, the constituents of family 3,

the kininogens have been reported to posses three domains, out of which two

participate in the inhibitory mechanism while one lacks any ability to inhibit the

proteinases. A typical cystatin domain was defined to be an approximately 100-amino

acids polypeptide that folds into a five stranded β-sheet, which partially wraps around

a central α-helix. [Fig.3] [Bode et al., 1988].

Later, the term ‘type’ was introduced and the mammalian cystatins were

divided into types 1, 2 and 3 [Rawlings and Barrett, 1990]. However, an increasing

number of cystatins from various sources introduced new subdivision of the cystatins

into four families [Rawlings and Barrett, 1990], the fourth family consisting of non-

inhibitory homologues of two cystatin-like domains, such as human α2 SH-

glycoprotein (feutin) and histidine-rich glycoprotein [Brown and Dziegielewska

1997]. The cystatin superfamily also comprises of phytocystatins. According to

recently proposed classification of peptidase inhibitors into families and clans

[Rawlings et al., 2004] cystatins are assigned to family I25, which consists of three

subfamilies, I25A (stefins), I25B (cystatins), I25C (are mostly not proteinase

inhibitors). Table 1 presents this classification with some examples.


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TABLE-1: CLASSIFICATION OF CYSTATINS AND OTHER

CYSTEINE PROTEINASE INHIBITORS AND THEIR

EXAMPLES

Group I: Traditional cystatin superfamily

1. Stefins Stefin A

2. Cystatins Cystatin C

3. Kininogens HMW Kininogen

4. Phytocystatins Rice bran cystatin

Group II: Structurally cystatin-like molecules with no inhibitory

activity Histidine rich glycoprotein (HRG) Human HRG

Fetuins Human fetuin

Group III: Cysteine proteinase inhibitors with no structural

homology to group I Thyropins Equistatin

Serpins Chagasin

Clitocybin Mycocypins


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Figure 2: Diagrammatic representation of the chain structure of

proteins in the cystatin superfamily

Type I is stefin, type II is cystatin and type III represents kininogens.

The structure indicated for kininogens is that of L-kininogen with

shorter C terminal; H-kininogens have a longer carboxyl terminal

extension. The Marks ( ) show the potential sites for the attachment of

the carbohydrate side chains.


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Figure 3: Folds of cystatin C

Cystatin C chain trace is shown in orientation which positions the N-

terminal “elephant trunk”, the first and the second hairpin loops to the

bottom from left to right [Adapted from Bode et al., 1988, EMBO J,

2593-2599].

N Terminal


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1.7 GENERAL PROPERTIES OF THE CYSTATIN SUPERFAMILY

Family 1 (Type I Cystatins): Stefins

The type I cystatins belong to the subfamily I25A.The protein inhibitors

belonging to the stefin family are single chain proteins which lack disulphide bonds

and carbohydrates and are composed of ≈100 amino acid residues with MW of about

~11 kDa. They are primarily intracellular cytoplasmic proteins of many cell types,

although they have been found in extracellular fluids as well [Abrahamson et. al.,

1986]. All type 1 cystatins have blocked N-terminal and contain methionine

representing the points of initation of translation [Turk et al., 1995a]. Recently a

highly versatile and roboust scaffold STM (Stefin A triple mutant) has been derived

from stefin A. The scaffold is amenable to engineering at multiple locations

[Hoffmann et al., 2010]. There are three important member of this family; cystatin A

and B isolated from human skin [Green et al., 1984; Lenney et al., 1979] and stefin C

purified from bovine thymus [Turk et al., 1986]. Cystatins α and β are considered to

be simply the rat species variants of cystatin A and B [Lenney et al., 1979].

Cystatin A

It is an inhibitor of cathepsin B in human skin discovered by Fraki [1976].

Later on Jarvinen [1978] studied it as ‘acid cysteine proteinase inhibitor’ (ACPI)

because of its acidic pI at 4.7-5.0. Brzin et al. [1983] purified an inhibitor from blood

leucocytes, and named it as “stefin’. The amino acid sequence was determined by

Machleidt et al. [1983]. Green et al. [1984] characterized same type of CPI from

human liver and latter renamed it as cystatin A. Cystatin A occurs in multiple

isoelectric forms with predominantly acidic pI values in the range 4.5-5.0 [Hopsu-

Havu et al., 1985]. Rinnie et al. [1978] detected cystatin A in extracts of squamous

epithelia from oesophagus. It was also found in dendritic reticulum cells of the lymph

nodes [Rinnie et al., 1983], seminal plasma [Minakata and Asano, 1985], saliva,

bovine skin [Turk et al., 1995] and human nails [Tsushima, 1993]. Cystatin A has

found to be a sensitive marker during HIV-I infection and for regeneration of

follicular lymphoid tissue [Voltersvik et al., 2006].


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Cystatin α

It is assumed to be a species variant of cystatin A found in rats. This protein

was characterized by Jarvinen [1976] as a specific inhibitor of CP from rat skin

having MW of 13 kDa. Cystatin α is generally found on the epidermal layer [Jarvinen

et al., 1978] and various other squamous epithelia [Rinnie et al., 1978]. Human stefin

A is expressed at high levels in skin and presumably controls cysteine proteinases in

the skin. Some cathepsins play a crucial role in the antigen presentation process

indicating that the interactions between stefin A and cathepsins contribute to the

species dependent diversity of the endosomal compartments which participate in the

immune response [Mihelic et al., 2006].

Cystatin B

Cystatin B was detected as an inhibitor of cathepsin B and H in human tissues

by Lenney et al. [1979]. Jarvinen and Rinnie [1982] purified cystatin B from human

spleen. Green et al. [1984] purified it from human spleen and liver with separation of

multiple forms. Cystatin B is a relatively basic protein with pI values of 6.25 and 6.35

for the two forms of cystatin B [Green et al., 1984]. Human cystatin B forms dimmer

but dimeric form does not show inhibitory activity [Green et al., 1984; Jarvinen and

Rinnie, 1982]. With ubiquitous distribution it appears to be general inhibitor in the

cytoplasm.

Cystatin β

The existence of a thermostable and dialyzable inhibitor of cathepsins B, H

and dipeptidyl peptidase I in the supernatant of rat liver homogenate was reported by

Finkelstadt [1957] and Lenney [1979]. The pI values of cystatin β ranges from 5.04 to

5.6 [Kominami, 1981]. Cystatin β has an almost even distribution in the tissues and is

more abundant than cystatin α in all tissues expect skin. This characteristic of cystatin

β resembles cystatins B of human variant.

Stefin C

Stefin C is a novel type of inhibitor from the stefin family. This inhibitor

exhibits considerable sequence homology with all type B stefins and especially with

bovine stefin B, it differs from the latter by its prolonged N terminus, the presence of


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a tryptophan residue, the occurrence of only one methionine on the N terminus instead

of 2 in stefin B and the presence of Leu instead of Cys [Turk et al., 1993a]. The

protein consists of 101 amino acid residues and its MW is calculated to be 11,546.

The inhibitor was found to be acidic with pI values from 4.5 and 5.6 [Turk et al.,

1993a].

Family 2 (Cystatins): Cystatins

Cystatins are typically about 120–125 residues long and contain four cysteine

residues, which are involved in the formation of the two disulfide bonds characteristic

of the family. Their molecular weight is a bit higher than that of stefins, about 13 kDa

[Vray et al., 2002]. Cystatins of class 2 are non-glycosylated with the exception of the

rat cystatin C [Vray et al., 2002], cystatin F and cystatin E/ M. Some cystatins are also

phosphorylated, but this phosphorylation does not affect their inhibitory activity

[Gerhartz et al., 1997; Laber et al., 1989]. They are synthesized with the signal

peptide and usually occur in secretions like chicken egg-white and other fluids

including blood plasma [Barrett et al., 1986]. The known members of this family are

chicken cystatin, human cystatin C, D, S, SN, SA, E, F and cystatin M.

Chicken Cystatin

Chicken egg white cystatin was the first family 2 cysteine proteinase inhibitor

to be isolated. It was first isolated from chicken egg white by Fossum and Whitaker in

[1968] and by Sen and Whitaker in (1973) who reported it as low MW tight binding

CPI of ficin and papain and of cathepsin B and C [Sen and Whitaker, 1973].It forms a

tight, reversible 1:1 complex with most known cysteine proteinases [Nicklin and

Barrett, 1984]. Its amino acid sequence was determined independently by Turk et al.,

[1983] and Schwabe et al. [1984] it consists of 116 residues with molecular weight of

14 kDa. It occurs in two major isoelectric forms (form 1, pI = 6.5, and form 2,

pI = 5.6).

Chicken cystatin has also been detected in the serum of both male and female

chickens at a concentration of 80µg/ml in egg white and 1µg/ml in the serum

[Anastasi et al., 1983]. It has also been found in chicken muscle cells [Wood et al.,

1985] and showed resemblance to cystatin C. Chicken cystatin has been shown to


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alter intracellular proteolytic processing of poliovirus proteins, resulting in a reduction

of virus yield [Korant et al., 1985]. These investigators suggested that the cystatin is

able to penetrate in the cellular cytoplasm and inhibits the action of a poliovirus-

coded proteinases. As a result, cystatins or their derivatives are being considered as

potential antiviral agents [Korant et al., 1988].

Human Cystatin C

Cystatin C was formely known as γ-trace or post globulin. It was first

described in 1961 as a constituent of urine from patients with renal tubular failure and

normal cerebrospinal fluid [Butler and Flynn, 1961 and Clausen, 1961].When γ trace

was shown to inhibit cysteine proteinases; it was renamed as cystatin C [Barrett et al.,

1984]. It is reported that γ-trace is a component of amyloid fibrils in patients suffering

from hereditary cerebrovascular amyloidsis, thus indicating its very important

biological role in metabolic processes [Cohen et al., 1983].The crystal structure of

human cystatin C reveals that the protein refolds to produce very tight two-fold

symmetric dimmers while retaining the secondary structure of the monomeric form.

The dimerization occurs through three dimensional domain swapping, a mechanism

for forming oligomeric proteins [Fig.4]. In the case of dimerization, the two

interacting molecules reconstitute the two monomeric topologies in a symmetric

fashion, as shown in Fig. 4. The energetic advantage of the symmetric dimer may be

related to the formation of the strong β-sheet interactions at the ‘open interface’.

Unhampered chain-like oligomerization could start with the reconstruction of only

one α–β domain, leaving the other two α- and β-structures available for interactions

with additional monomers [Janowski, 2001].

Besides, the intact molecule, several peptide derivatives of cystatin C have

been shown to inhibit cysteine proteinases. They include a synthetic peptide (Z-Leu–

Val-gly-C-NH2) that mimics the proposed binding centre of cystatin C. This peptide

was also shown to have antibacterial activity by specifically blocking the growth of

streptococci both in vitro and in vivo [Bjorck et al., 1990]. Cejka and Fleischmann

[1973] determined the isoelectric point of cystatin C to be 9.0 although; values

ranging from 8.0 to 9.5 have also been reported [Brzin et al., 1984].


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Figure 4: Schematic illustrations of human cystatin C (HCC)

oligomer formation. a. Two monomers form a dimer

b. Three-dimensional domain swapping.

c. In an open-ended variant, the same mechanism may lead to

cross β-fibril structure. In this diagram, the cystatin fold is

represented by an α-helix (cylinder) running across the concave

face of a β-sheet (stripes). In a screw operation, new components

are added by rotation followed by a translation along the screw

axis. [Adopted from Janowski et al., 2001, Nature Structural

Biology, 316-320]


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Cystatin D

Freiji et al. [1991] cloned a new member of human cystatin multigene family

from a genomic library using cystatin C cDNA probe. This inhibitor consists of 122

amino acids residues having a MW of 13,885. The deduced amino acid composition

includes a putative signal peptide and has 51- 55% homology with either cystatin C or

secretory gland cystatins S, SA and SN. It is a relatively neutral protein with pI in the

range of 6.8- 7.0 [Freiji et al., 1991]. It is expressed in parotid glands, saliva and tears

[Balbin et al., 1994]. This tissue restricted expression is in marked contrast with a

wider distribution of all other family 2 cystatins.

Cystatin S

Human saliva contains several low MW acidic proteins which include cysteine

proteinase inhibitor [Isemura et al., 1984b].The first salivary inhibitor purified and

sequenced was SAP-I [Isemura et al., 1984a], which was renamed as ‘cystatin S’. It

was found to have 54 and 41% sequence homology with cystatin C and chicken

cystatin, respectively [Isemura et al., 1984b]. This inhibitor has also been isolated

from human submaxillary, submandibular and sublingual glands and found to be

present in the serous cells of the parotid and submaxillary glands [Isemura et al.,

1984b]. The protein has also been found in tears, serum, urine, bile, pancreas and

bronchus [Isemura et al., 1986]. Cystatin S contains no phosphate, in contrast to other

salivary proteins.

1.8 VARIANTS OF CYSTATIN-S

Several molecular variants of cystatin S have been studied by Isemura et al.

[1986]. They differ in their N-terminal sequences and pI values [Minakata and Asano

1985; Isemura et al., 1986]. Differences in pI values resulted from phosphorylation of

residues Ser 3 and Ser 1 in salivary glands.

Cystatin SN

Originally known as cystatin SV or SA-1 [Abrahamson et al., 1986]. It

consists of 121 amino acid residues and MW value of 14,316. The pI is in the range of

6.6-6.8.


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Cystatin SA

The protein consists of 122 amino acid residues and MW value is slightly

higher than SN, which is 14,351. It has acidic pI value of 4.6 [Isemura et al., 1991].

Cystatin SA isolated from saliva had N-terminal residue Glu.

Cystatin E

Cystatin E was first reported by Ni et al. [1997] by expression of amniotic

fluid and fetal skin epithelial cell cDNA libraries. The mature protein is a polypeptide

of 121 amino acid residues and has 28 residue signal peptide having a MW of 15,000.

Cystatin E, resembles family 2 cystatins structurally in containing two protective

disulphide bridges and by being a secreted protein. The inhibitor has unusual

characteristic of being a glycoprotein carrying an N-linked oligosaccharide at Asn 108

[Stenman et al., 1997].Cystatin E has been detected in a variety of specialized tissues

and organs [Ni et al., 1997]. Its high concentration is found in uterus, liver and lower

but significant amounts in placenta, pancreas, heart, spleen, small intestine and

peripheral blood leucoytes, in brain, testis and kidney of mammals. The inhibitor

serves a protective role during fetal development.

Cystatin F or Leukocystatin

Leukocystatin (also called cystatin F) is a cystatin recently discovered and

mainly found in human natural killer cells, in dendritic cells derived from activated

stem cells and murine T cells. Cystatin F is the only cystatin synthesized and secreted

as an inactive disulphide linked – dimeric precursor [Cappello et al., 2004]. Following

reduction to the monomeric form cystatin F becomes active [Langerholc et al., 2005]

and found to inhibit cathepsins F, K, L and V strongly and to a lesser extent,

cathepsins S and H [Ni et al., 1998]. It was shown that a major target of cystatin F in

various immune cell types is cathepsin C that activates serine proteinases in T-cells,

natural killer cells (NK), neutrophils and mast cells [Hamilton et al., 2008]. It is

important to note that cystatin F is a glycosylated cystatin.


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Cystatin M

Cystatin M was discovered in the primary tumor cell line. It is synthesized as a

precursor protein and is secreted in the same form [Heijne, 1985]. The predicted

molecular mass is approximately 14.3 kDa and pI of 7.8 for the mature protein. An N-

glycosylated form of cystatin M of 20-22 kDa was co-immunoprecipitated and

accounted for about 30-40% of total cystatin M protein. Both forms of native cystatins

of this class M occurred intracellularly. Cystatin M resembles other members of the

family in having the three conserved domains. It has 40% homology between cystatin

E and other cystatins. The homology range from 30-40% for conserved amino acid

residues and 25 to 33% for identical amino acids. Cystatin M shows the closest

homology with cystatins C, they share 33% identical and 38% conserved amino acid

residues [Sotiropoulou et al., 1997].Cystatin M is expressed in normal tissues but

expression is lost in most late stage/metastatic breast cancers [Rivenbark et al.,

2006]. However, increased cystatin M expression was shown to decrease tumor

invasion, proliferation and adhesion to endothelial cells [Shridhar et al., 2004].

Cystatin M silencing with and RNAi approach in an oral cancer cell line not

only increased cell invasion and motility but also increased cell proliferation by an

unknown mechanism [Vigneswaran et al., 2006]. In contrast, through laser capture

microdissection of breast cancer cell, cystatin M (and C) correlated positively with

tumor size but not with metastatic activity [Vigenswaran et al., 2005]. Vigneswaran et

al. [2003] also described elevated cystatin M in metastatic squamous cell carcinoma.

It has been shown that cystatin M is frequently epigenetically inactivated during

breast carcinogenesis and cystatin M expression suppresses the growth of breast

carcinoma [Schagdarsurengin, 2007].

Family 3 (Type III Cystatins): Kininogens

Most complex cystatin molecules are those of the type 3 cystatins, the

kininogens are found only in plasma. Kininogens have been known for a long time as

the precursors of kinin. There are three distinct types of kininogens, designated as

high molecular weight kininogen (H-kininogen HMWK) with MW of about 120000,

low-molecular weight kininogen (L-Kininogen LMWK) with MW of about 68000-

70000 and a structurally different T-Kininogen (also known as “major acute-phase


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protein” with MW of about 68000 [Barrett, 1986a; Barrett, 1987; Muller-Esterl, 1989]

only in rat plasma.

They are all single chain proteins consisting of two-chain forms, a heavy and a

light chain. Proteolysis of kininogen by kallikreins releases kinin segment and

converts it into two chains. The various types of kininogen have the same basic

structure, a heavy chain of 50-60 kDa at the amino terminus, the kinin segment in the

core part of the molecules and a light chain of variable length at the carboxy-terminus

[Kitamura et al., 1985; Lottspeich et al., 1985].

The primary structure of the heavy chain, kinin portion and a segment of 12

amino acid residues are identical in HMWK and LMWK, but the structure of their

light chain diverges considerably. HMW has a large light chain of 45-58 kDa which

harbours unique histidine-rich region [Sugo et al., 1980]. The HMW kininogen light

chain contains the binding site for prekallikrein and factor XI. The kininogens (L and

H) are strong inhibitors of papain and cathepsins L and weak inhibitors of cathepsin H

and particularly cathepsin B [Barrett, 1986a; Muller-Esterl, 1985; Barrett, 1987].

T-kininogen (also called as thio statin) found only in rats is an exception to

the general rule for kininogens, since it is not susceptible to kallikerin hydrolysis.

However, cathepsin D can liberate T-kinin (Ile-Ser bradykinin) from T-Kininogen

[Okamoto et al., 1983; Sakamoto, 1988]. Strong inhibitory capacity against papain

and cathepsin L with T-kininogen has been reported by Moreau [1989].

Plasma concentrations of kininogens have been determined by radio immune

assay to be 109-217µg/ml (L-kininogens) and 69-116 µg/ml (H-kininogen) [Adam et

al., 1985]. Kitamura et al. (1985) and Salvesen et al. (1986) found that three cystatins

like segements are present in kininogens, although the first lacks the preserved

sequence Gln-Val-Val-Ala-Gly. It is known that the LMW-kininogen contain no free

thiol groups [Salvesen et al., 1986; Kitamura, 1985]. It has been shown that

L-kininogen binds two molecules of papain, cathepsins S and L with high affinity

[Turk et al., 1995b]. Similarly, H-kininogen binds two molecules of papain, cruzipain

and cathepsins S [Turk et al., 1996]. Like the type 2 cystatins, both inhibitory

domains of LMWK and HMWK are grouped in subfamily I25B of the cystatin

superfamily [Rawlings et al., 2004].


20

1.9 NEW MEMBERS OF THE CYSTATIN SUPERFAMILY

The feutins and histidine-rich glycoproteins (HRG) comprise fourth family

of cystatins. The feutin family consists of two tandem cystatin domains. Bovine feutin

was first characterized by Pedersen in 1944, and its relation to cystatin superfamily

described in 1988 [Elzanowski et al., 1988]. Human feutin (α2-HS glycoprotein) was

confirmed in 1987 [Dziegielewska et al., 1987; 1990; Dziegielewska and Brown,

1995]. Since then, protein and/or cDNA sequences have been reported for human,

cow, pig, rat, mouse, habu snake, feutins [Brown and Dziegielewska, 1997]. Almost

all the feutin sequences contain 12 cysteine residues, showing homology to the

cystatins and cystatin domains in kininogens [Dziegielewska and Brown, 1995]. HRG

has been characterized in the plasma of man, mouse, rabbit, cow and pig [Leung,

1993], sharing good sequence homology with human and bovine HMW kininogen

[Koide et al., 1986]. A large number of proteins have been discovered recently, which

possess cystatin domains and may even exhibit cysteine proteinase inhibitory (CPI)

activity e.g. latexin [Aggarwal et al., 2007]. However, feutin, HRG and latexin all

seem to lack cysteine proteinase inhibitory activity.

1.10 OTHER CYSTATINS AND CYSTATIN RELATED

PROTEINS

CRES Protein

There are a number of other cystatins or cystatin related proteins, expressed in

different tissues and cell types in humans and other mammals. A novel cystatins-

related epididymal specific (CRES) gene was found in mouse epididymis, showing

substantial homology with those of well established protein inhibitors, cystatins

[Cornwall, 1992]. However the CRES gene does not contain the highly conserved

QXVXG region and P-W pair which are crucial for cathepsin inhibition. The CRES

gene is almost restricted and much less expressed in testis, and no expression in any

other tissue was found. A gene was isolated from mouse foetus, related to the genes

that encode cystatins, and was named testatin [Tohonen et al., 1998]. Testatin

expression is restricted to pre-sartoli cells and its expression was high during the early

events of testis development. Two more genes from sartoli cells, named cystatin SC


21

and cystatin TE-1, were isolated which were detected only in the testis and highly

expressed in testis and epididymis, respectively [Li et al., 2002]. The role of this

subgroup of cystatins might be regulation of proteolysis in this reproductive tract as

well as protection against invading pathogens by inhibiting microbial proteinases, as

shown by cystatin 11 [Hamil et al., 2002].

Tick Cystatins

Genes encoding cystatins have also been found in several ticks which

constitute the main vector of Lyme disease in the USA and Europe. The two cystatins

transcripts are encoded by two different genes in the tick Ixodes Scapularis. Both

cystatins were expressed and were named as sialostatin L [Kotsyfakis et al., 2006;

Kotsyfakis et al., 2007]. The name indicates the strong inhibition of cathepsin L.

These two sialostatins, which were found in saliva, show 75% identity in their

sequence and inhibit almost equally cathepsin L, with Ki =4.7 nM, and cathepsin V

with Ki =57 nM. Cathepsin L, is a known collagenolytic enzyme and plays an

important role extracellularly and intracellularly. Its collagenolytic activity is

inhibited by chicken cystatin [Maciewicz et al., 1987]. Consequently, sialostatin L

displays an anti-inflammatory role and inhibits proliferation of cytotoxic T-

lymphocytes [Kotsyfakis et al., 2006].

Staphostatins

These are newly described family whose members are the specific inhibitors

of staphylococcal cysteine proteinases. Three members of this family have been

described – staphostatins A and B from Staphylococcus aureus and staphostatin A

from Staphylococcus epidermidis [Filipek et al., 2003].

Phytocystatins

In plants, inhibitors of cysteine proteinases are known as phytocystatins. They

contain the QXVXG region of type 2 cystatins, but also resemble stefins in the

absence of disulphide bonds [Arai et al., 2002], providing a transitional link between

type 1 and type 2 cystatins. There are numerous phytocystatins expressed and

characterized for the protein level from corn [Abe et al., 1992], rice [Chen et al.,


22

1992], soyabean [Lalitha et al., 2005] and sugarcane [Oliva et al., 2004]. C-terminal

extended phytocystatins were found as bifunctional inhibitors of papain and legumain

[Martinez et al., 2007]. In addition, a “multicystatin” containing two cystatin like

domains were isolated from cowpea leaves [Diop et al., 2004] and tomato leaves [Wu

and Haard 2000]. Also there are certain plant proteins like monellin which lack the

cysteine proteinase inhibitory activity but have a cystatin like three dimensional

structure [Grzonka et al., 2001]. Phytocystatins and other inhibitors are important for

plant defence response to insect predation, it may act to resist infection by some

nematodes [Koiwa et al., 1997] play a crucial role in response to various conditions

[Diop et al., 2004; Brzin and Kidric, 1995] and show great potential tools for

genetically engineered resistance of crop plants against pests [Aguiar et al., 2006].

1.11 NON SPECIFIC CYSTEINE PROTEINASE INHIBITORS OF

THE PAPAIN FAMILY

A number of other proteins exhibt inhibitory activity against lysosomal

papain–like cysteine proteinases. α-2 macroglobulin unspecifically trap

endopeptidases of different types, blocking the access of protein substrates to the

active site of the trapped proteinases without inactivating them [Armstrong, 2001].

Thyropins

Thyropins share considerable sequence homology with the thyroglobulin type

1-domain present in eleven copies in the prohormone thyroglobulin and in a number

of other proteins from other organisms [Molina et al., 1996]. Cathepsin L is

specifically inhibited by a mammalian representative of this class, the major

histocompatibility complex (MHC) class II –associated p41 invariant chain fragment

[Bevec and Garver, 1996, Guncar et al., 1999].

Serpins

Serpins as typical protein inhibitors of serine type proteinases can also inhibit

cysteine-type proteinases including papain-family of cysteine type proteinases in

cross-inhibition [Turk et al., 2002]. This was demonstrated for the human squamous

cell carcinoma antigen1 (SCCA) as a potent inhibitor of cathepsins K, L and S129, its


23

mouse ortholog SQ-N-5 which inhibits an addition cathepsins V but not cathepsin B

and H [Schick et al., 1998].

Chagasin from trypanosome cruzi has been shown to inhibit a number of

papain-like proteinases [Monteiro et al., 2001]. Chagasin-like proteins were found to

be encoded by genomes of several eukaryotes, bacteria and archea, and thus appears

to be the first protein inhibitor of cysteine proteinases identified in prokaryotes

[Ridgen et al., 2002]. Chagasin is the only proteinase inhibitors known that adopt an

immunoglobulin type fold [Rigden et al., 2001]. In addition, α-2 macroglobulin is

known as the only protein inhibitor that can inhibit several different types of

proteinases, including the papain family of cysteine proteinases [Mason, 1989].

Clitocybin is a new type of cysteine proteinase inhibitor from a mushroom.

Clitocybin appears to be related to fungal lectins and the team that found it suggested

a new family of cysteine proteinase inhibitors called mycocypins [Brzin et al., 2000].

1.12 VARIANTS IN CYSTATINS SUPER FAMILY

Divergent cystatins showing significant homology to stefins, cystatins and

kininogens have been expressed/purified and characterized from venom of African

puff adder (Bitis arietans) [Evans and Barrett, 1987]; from perilymph of flesh fly

larvae [Suzuki and Natori, 1985]; from Drosophila melanogaster [Delbridge and

Kelly, 1990], goat brain [ Sumbul and Bano, 2006 ], goat lung [Khan and Bano, 2008]

goat pancrease [ Priyadarshini and Bano 2010]. Some of the mammalian and non

mammalian sources from where cysteine proteinase inhibitors (CPIs) have been

isolated are summarized in Table 2.

1.13 EVOLUTION

The first two proposed evolutionary dendrograms for CPIs were made based

on a small number of members of the cystatin superfamily [Muller-Esterl et al., 1985;

Salvesen et al., 1986]. The new proposed evolutionary dendrograms followed the

evolution of the proteins of the cystatin superfamily along four lineages, with special

attention that duplication of cystatin like segments has played important contribution

to the understanding of the evolution of cystatins.


24

TABLE- 2: LOW AND HIGH MOLECULAR WEIGHT CPIs

FROM DIFFERENT SOURCES

Source Tissue MW pl Reference

African puff Adder 13,000 6.5 Evans and Barrett, 1987

Beef Spleen 13,000 4.8-7.0 Brzin et al., 1982

Bovine Brain 25,000 4.7 Aghajanyan et al., 1988

11,000 5.23

Hoof 11,406 - Tsushima et al., 1996

Muscle 14,000 6.2 Bige et al., 1985

14,300 Zabari et al., 1993

Colostrums 12,787 10.0-10.3 Hirado et al., 1984

Dog Colostrum - - Poulik et al., 1981

Parotid gland

& Kidney Sekine and Poulik, 1982

Guinea pig Skin - Acidic Jarvinen, 1976

Horse shoe crab Hemocytes 12,600 - Agarwala et al., 1996

Hog Kidney - - Lenney et al., 1979

Human Liver 12,400 - Green et al., 1984

Spleen 11,400 4.7-50 Jarvinen and Rinnie, 1982

Placenta 12,500 - Rashid et al., 2005a

Rabbit Liver 5000-

10,000 - Pontremoli et al., 1983

Skin 12,500

12,500 6.6 Hayashi, 1975

Rat Brain - - Kopitar et al., 1983

Trypanosoma

Cruzi - 11,000 - Monteiro et al., 2001

Goat

Brain 70,800

12,720 Sumbul and Bano, 2006

Pancreas 44,000 Priyadarshini and Bano, 2010

Kidney 67,000 Zehra et al., 2005

Lung 66,400

76,400 Khan and Bano, 2009a

Muscle 58,800 Aatif and Bano, 2011

Liver 38,200 Shah and Bano, 2011


25

According to the scheme of Muller-Esterl et al. [1985] constructed on the

basis of sequence homology, the diversity of CPI has evolved from two ancestral

building blocks ‘A’ and ‘B’ [Fig. 5]. The stefin progenitor represents the whole

superfamily comprising a single ‘A’ unit. Cystatin acquired a second element B,

possibly by gene fusion, thus forming ‘AB’ unit. Gene triplication of the archetype

inhibitor generated the kininogen heavy chain which contains 3 cystatin like copies

(AB)3. The proposed evolutionary pathway also contained a ‘missing link’, a two

cystatin domain protein that evolved from the cystatins by duplication, with two

candidates for such a protein: feutin and HRG. This scheme however seems unlikely

most importantly because neither domain in feutins/HRG is inhibitory but two

domains of kininogens have inhibitory activity. If feutin/HRG were the ‘missing link’,

then the kininogens which have evolved from the two domain protein would have to

re-evolve their protease inhibitory activity and sequences [Brown and Dziegielewska,

1997]. Brown and Dziegielewska, [1997] proposed the following scheme for cystatin

superfamily evolution, with features similar to Muller-Esterl et al. [1985] scheme but

with a new missing link, a two cystatin domain protein in which both the domains

were functional cysteine proteinase inhibitors. From it, the kininogens, feutins, and

HRG could have evolved separately or perhaps in parallel and retained or lost their

protease-inhibitory activity and active site sequences. This scheme [Fig. 6] draws

support from the observation of conserved sequences immediately around the cysteine

at the C-terminus of the feutins, HMW-kininogens and HRG, again suggesting a

common origin for these three proteins. Based on this Lee et al. [2009] have recently

grouped feutins, HRG and kininogens in a single family, type 3 cystatins. Thus in the

cystatin superfamily, as in other known protein families, the common building blocks

(cystatin domains) have been used to create functionally diverse proteins.

1.14 STRUCTURE OF CYSTATINS

Primary Structure

Most of the members of cystatin superfamily are polypeptides of 98-126

amino acid residues with MW values in the range of 11-14kDa. As regards to the

amino acid composition of cystatins few distinctive features can be attributed to the

subfamilies.Stefins are devoid of disulphide linkages (human cystatin A and rat

cystatin α lack cysteine residues while human cystatin B and rat cystatin β have 1 and


26

Figure 5: Evolution of cystatin superfamily. A schematic

presentation of protein structure in cystatin superfamily

by Muller-Esterl et al. [1985]

Unit A is archetypal cystatin and contains conserved QVVAG

sequence. Unit B has two disulfide bonds. The heavy chain of

kininogen consists of three cystatin domains (AB)3 and the light chain

of kininogen can be shorter or longer thus determining LMW or HMW

kininogen. (□) small box represents disulphide bonds.


27

Figure 6: Evolutionary scheme proposed by Brown &

Dziegielewska [1997] for the three types of cystatins.

The evolutionary relationship between all known inhibitory

active human cystatin families. D2 and D3 represent the domain

2 and domain 3 of kininogens.


28

2 cysteine residues, respectively) and tryptophan. Turk et al. [1993b] however

reported the presence of tryptophan in stefin C also. A unique feature of stefin B is the

conserved QVVAG region in the stefins of mammalian origin, with Val 54 replaced

by Leu 54. Ni et al. [1998] reported the presence of an additional disulphide bridge in

cystatin F, for stabilizing the N-terminal part of the molecule in addition to the

presence of second tryptophan residue, along with the conserved Trp 106,

characteristic of type 2 cystatins. The alignment of sequences of cystatins reveals

common features, significant to the structure and activity of the proteins. Four

residues are common to all the sequences of cystatins and inhibitory kininogen

segments: Gly9, Gln53, Val55 and Gly57. These residues are considered to be of

functional importance since they are absent from the non-inhibitory segment D1 of

kininogens. Another six conserved residues are Val47, Val55, Ala56, Tyr60, Cys71

and Tyr100. The segment Gln53 to Gly57 is the most highly conserved region.

Secondary Structure

The structures of chicken cystatin and human cystatin C have been studied

extensively. Based on the lines of evidences from analysis of amino acid sequence for

the distribution of hydrophilic and hydrophobic residues, the predicted hydrophilic

residues are considered to be on the surface whereas hydrophobic regions are internal

like other compact globular proteins. Each sequence possesses a large N-terminal

hydrophilic region (15-42 residues) and a prominent central hydrophobic region

(55-70 residues) followed by another large hydrophilic region at C-terminus. The

secondary structure of human cystatin C in solution is very similar to that reported for

chicken cystatin [Dieckmann et al., 1993]. The crystalline form of chicken cystatin

has been reported by Bode et al. [1988].

Both human cystatin C and chicken cystatin as well as the family 1 cystatins

(A and B) [Martin et al., 1994; Stubbs et al., 1990] consist of five stranded

antiparallel β-pleated sheets which is wrapped around a straight five turn α-helix. An

appending segment of partial α-helical geometry is present in chicken cystatin

[Saxena and Tayyab, 1997] which was not found in human cystatin C by X-ray

diffraction [Bode et al., 1988].The conformation of this region seems to be closer to

that found for chicken cystatin in solution which consists of two loops [Dieckmann et


29

al., 1993]. The N-terminal segment up to Val 10 is flexible, similar to that of other

cystatins [Martin et al., 1995]. Trp was found only in the second hairpin loop of

cystatins [Bode et al., 1988]. A unique feature was observed in crystal structure of

cystatin F in its dimeric ‘off’ state. The two monomers interacted in a fashion not seen

before for cystatins or cystatin like proteins, crucially dependent on an unusual

intermolecular disulphide bridge. The core sugars for one of the two N-linked

glycosylation sites for cystatin F are well ordered and probably their conformation

and interactions with the protein modulate its inhibitory properties in particular its

reduced affinity towards asparaginyl endopeptidase compared with other cystatins

[Schuttelkopf et al., 2006].

The three dimensional structure of stefin A and B is shown in Fig. 7 A and

7 B. Stefins have a well defined globular fold consisting of five antiparallel β strands

wrapped around a central five turn α helix. There is considerable similarity between

the structural features of stefins A and B but there are also some important differences

in the region which are fundamental to proteinase binding. The differences consist

primarily of two region of high conformational heterogenity in free stefin A which

corresponds in stefin B to two of the components of the tripartite wedge that docks in

to the active site of target proteinases. These regions which are mobile in solutions are

the five N-terminal residues and the second binding loop. In the bound conformation

of stefin B they form a turn and a short helix, respectively.

Circular dichroism and computer prediction of secondary structure from the

sequence indicates that the chicken cystatin has about 20% α-helix, 42% β-structure,

and 24% β-turn and 12% random coil [Schwabe et al., 1984]. Janowski et al., [2001]

proposed that human cystatin C undergoes dimerization under mild denaturating

conditions. NMR studies revealed that the majority of the proteins are not affected by

dimerization, however, the cysteine proteinase binding site is significantly affected

[Ekiel et al., 1997] which accounts for the activity loss [Abrahamson, 2001]. Hence, it

can be concluded that hydrophobic CP binding site is involved in the monomer-

monomer contact. The dimerization may be relevant to regulation of cystatin C

inhibitory activity in vivo [Abrahamson, 2001]. Three dimensional swapping studies

indicate the dimerization of human cystatin C to be a step towards protein

oligomerization [Janowski et al., 2001].


30

(A)

(B)

Figure 7: Three dimensional structures of stefin A and B

Ribbon representation of the minimized average structure of stefins,

illustrating the 5-stranded antiparallel β sheet wrapped around the central

α-helix with the C-terminal loop running along the convex face of the

sheet.

(A) Structure of stefin A

(B) Structure of stefin B


31

1.15 INHIBITION OF PROTEINASES

Specificity

Cystatins are highly specific for CPs except for thyropins which shows

inhibitory activity against aspartic and metalloproteinases [Mihelic and Turk, 2007;

Lenarcic and Turk, 1999]. However there are few cystatins capable of inhibiting

mammalian legumain [Alvarez-Fernandez et al., 1999] and calpains [Crawford et al.,

1987]. To date, none of the cytoplasmic inhibitors have been tested on ubiquitin

processing and recycling proteinases [Keppler, 2006]. Stefin A and B are potent

inhibitors of papain, cathepsin L, S and H but have decreased activity against

cathepsin B [Musil et al., 1991]. Type 2 cystatins are important endogenous inhibitors

of papain like CPs including cathepsins, parasite proteinases like cruzipain and

mammalian legumain [Turk et al., 2005; Turk and Bode, 1991]. HCC and chicken

cystatin inhibit papain, cathepsin L and S [Abrahamson et al., 2003]. HCC shows

strong inhibitory capacity for rapid binding thus neutralizing protease activity in an

emergency inhibition [Turk et al., 2005]. It also inhibits cruzipain, suggesting its

possible defensive role after infection [Stoka et al., 1995]. Cystatin F inhibits

cathepsin F, K, V, S, L and H [Langerholc et al., 2005] and weakly legumain

[Alvarez-Fernandez et al., 1999]. More recently it was found that the intracellular

form of cystatin F, after N-terminal truncation of the first 15 residues including

cysteine, inhibits cathepsin C [Hamilton et al., 2008]. Cystatin D inhibits cathepsin S,

H and L but not cathepsin B or pig legumain [Alvarez-Fernandez et al., 2005].

Human cystatin E/M inhibits papain, cathepsin B, L, V and legumain [Ni et al., 1997;

Sotiropoulou et al., 1997; Cheng et al., 2006; Alvarez-Fernandez et al., 1999].

Clostripain (protease not belonging to papain family) is also inhibited by cystatins

[Barrett et al., 1986].

1.16 KINETIC BEHAVIOUR

Cystatins are the first group of protein inhibitors of CPs for which the

mechanism of inhibition was investigated. All the cystatins are non-covalent,

competitive, reversible, tight binding inhibitors which inhibit the target enzymes in

micromolar to picomolar range [Turk et al., 1997]. They form tight equimolar


32

complexes with CPs [Anastasi et al., 1983]. Some of the reported values of

equilibrium constants for dissociation of complexes between human cystatins and

lysosomal CPs are summarized in Table 3. The affinity differences can be explained

by the differences in the active site regions of endo- and exopeptidases. The access of

the inhibitor to the active site of exopeptidases is partially obstructed by occluding

loops in cathepsin B [Musil et al., 1991] and cathepsin X [Guncar et al., 2000] and

propeptide parts in cathepsin H [Guncar et al., 1998] and cathepsin C [Turk et al.,

2001].

1.17 MECHANISM OF INTERACTION OF CYSTATINS WITH

PROTEINASES

It has been established that no disulphide bond is formed between the active

site cysteine and the inhibitor because complexes dissociate when denatured

with out reduction as was found with chicken cystatin [Nicklin and Barrett, 1984] and

kininogens [Gounaris et al., 1984]. Carboxymethylation of enzyme and inhibitor does

not prevent complex formation [Anastasi, 1983]. The active site of papain in complex

with chicken cystatin is unreactive with 5, 5’-dithio-bis (2-nitro benzoic acid) at pH

8.0, 2, 2-dipyridyldisulphite at pH 4.0 and [14C] iodoacetate [Nicklin and Barrett,

1984]. It has also been reported that the complex formation is accompanied by

spectroscopic changes, most likely reflecting local perturbation of the environment of

aromatic residues in both enzymes and inhibitor [Lindhal et al., 1998].

On the basis of cystatin domain structure, it was proposed that there are three

regions crucial for interaction with proteinases: the amino terminus and two β-hairpin

loops, one in the middle and one in the C-terminal segment of the protein. The first

loop contains a QXVXG sequence conserved in almost all inhibitory members of

cystatin, whereas the second loop contains a P-W motif, which is also highly

conserved. Both these loops and the amino terminus form a wedge shaped edge,

which is highly complementary to the active site of the enzyme. The N-terminally

truncated forms of chicken cystatin confirmed the crucial importance for the binding

of the residues preceding the conserved Gly-9 residue [Machleidt et al., 1989]. The

essential interactive elements of this hypothetical complex are shown in figure. 8.

Complex of stefin A interaction with cathepsin H is shown in figure 9.


33

Table 3: Equilibrium constants for dissociation (Ki) of complexes

between human cystatins and chicken cystatin with

lysosomal cysteine proteinases (human cathepsins, papain

and cruzipain).

Ki (nM)

Cystatin Papain Cathepsin B Cathepsin H Cathepsin L Cruzipain

Stefin A 0.019 8.2 0.31 1.3 0.0072

Stefin B 0.12 73 0.58 0.23 0.060

Cystatin C 0.00001 0.27 0.28 <0.005 0.014

Cystatin D 1.2 >1000 7.5 18 n.d.

Cystatin E/M 0.39 32 n.d. n.d. n.d.

Cystatin F 1.1 >1000 n.d. 0.31 n.d.

Cystatin S 108 n.d. n.d. n.d. n.d

Cystatin SA 0.32 n.d. n.d. n.d. n.d.

Cystatin SN 0.016 19 n.d. n.d. n.d.

Chicken cystatin

0.005 1.7 0.06 0.019 0.001

L-kininogen 0.015 600 0.72 0.017 0.041

H-kininogen 0.02 400 1.1 0.109 n.d.

n.d. (not determined), Ki values for human cystatins [Abrahamson et al., 2003],

chicken cystatin [Barrett et al., 1986] and cruzipain inhibition by cystatins [Stoka et

al., 1995].


34

Figure 8: Scheme of the proposed model for the interaction of

chicken egg white cystatin with papain

Interaction of chicken cystatin with papain has been illustrated in the fig.

(Turk and Bode, 1991, FEBS Lett, 213-219)


35

Figure 9: Three dimensional structure of the complex formed

between human stefin A and cathepsin H.

Ribbon representation of the crystal structures of the complex between

stefin A and cathepsin H (Turk and Turk, 2008, Acta chim Solv, 727-

738).


36

Bode et al. [1988] demonstrated that the major contribution is from the first

hairpin loop containing QVVAG sequence [Turk, 1985]. According to the model the

N-terminal segment of cystatin is flexible and bridges over the active site Cysteine 25

of papain without completely burying it. The side chain of Leu 8 binds to S2 subsite

of papain and determines the substrate specificity of papain [Asboth et al., 1988]. This

was supported by Brzin et al. [1984] who demonstrated that the truncated form of

HCC starting with Leu-Val before Gly-11 (corresponding to Gly-9 of chicken

cystatin) has virtually the same affinity for papain as the full length form whereas the

truncated form starting with Gly-12 has been reported to show 1000 fold weaker

inhibition [Abrahamson et. al., 1987]. However, Nycander and Bjork [1990]

emphasized the role of Trp-104 in the inhibition of cysteine proteinases. According to

their model, Trp-104 of cystatin interacts primarily with two Trp side chains in the

active side cleft of papain, Trp 177 and Trp 181, in such a manner that the indole ring

trp-104 stacks on the side chain of Trp 177 and the edge lies on the indole ring of Trp

181.

A two step mechanism of inhibition of the lysosomal cysteine proteinase

cathepsin B by its endogenous inhibitor cystatin C was observed by Nycander et al.

[1998]. An initial weak interaction in which N-terminal of the inhibitor binds to the

proteinase is followed by a conformational change. Subsequently, the occluding loop

of the proteinase that partially obscures the active site is displaced by the inhibitor

bringing about another conformational change. The presence of occluding loop of

cathepsin B renders it much less susceptible to inhibition by cystatin than other

proteinases. A similar two step binding of cystatin A to the cysteine proteinase was

suggested by Estrada and Bjork [2000]. The flexible N-terminal region of the cystatin

binds independently to the target proteinases after the binding of hairpin loops. It is

interesting that the replacement of the three N-terminal residues preceding the

conserved Gly of stefin A by the corresponding 10-residues long segment of cystatin

C increased affinity of the inhibitor for cathepsin B by about 15-fold [Pavlova and

Bjork, 2003], suggesting that the inhibitory potency of cystatin can be substantially

improved by protein engineering.

The crystal structure of human stefin A-porcine cathepsin H complex showed

small distortion of the structure upon formation of the complex [Jenko et al., 2003]. In


37

addition to the structurally derived data, the contribution of the individual residues

within protease binding region of cystatins was additionally investigated by

mutational analysis and kinetic studies performed by several different groups

[Auerswald et al., 1994; Estrada et al., 1999; Pavlova et al., 2000].

1.18 CYSTATINS: BIOLOGICAL ASPECTS AND PATHO-PHYSIOLOGY

Proteinases and their natural inhibitors may co-exist at different levels of

cellular evolution. Disturbing the harmony of the normal balance of enzymatic

activities of proteinases and their natural inhibitors may lead to severe biological

effects.

Cystatins constitute a powerful regulatory system for endogenous CPs which

are often secreted or leaked from the lysosomes of dying and diseased cells [Ekiel et

al., 1999]. They are known to play important roles in various pathophysiologic

conditions such as sepsis [Assfalg-Machleidt et al., 1988], cancer [Cox, 2009],

rheumatoid arthritis [Trabandt et al., 1991], purulent bronchiectasis [Buttle et al.,

1990], peridontitis [Lah et al., 1993], renal failure [Kabanda et al., 1995],

Alzheimer’s disease [Bernstein et al., 1996 ], multiple sclerosis [Bever et al., 1995],

muscular dystrophy [Sohar et al., 1988], etc. which indicate that a tight enzyme

regulation by cystatin is a necessity in the normal state.

Cystatins and cancer

Cathepsins are involved in the degradation of extracellular matrix, facilitating

the growth, invasion and metastasis of tumour cells and also in tumour angiogenesis

[Gocheva and Joyce, 2007; Mohamed and Sloane, 2006; Turk et al., 2004; Vasiljeva

et al., 2006]. Generally, cathepsin to cystatin ratio is found to be increased in most

tumour types compared to normal tissues [Paraoan et al., 2009; Rivenbark and

Coleman, 2009]. Elevated levels of TPIs in various tumour types have been correlated

to better prognosis like, stefin A positive breast cancer patients were much less likely

to develop distant metastasis [Parker et al., 2008], stefin A and B in non small cell

lung cancer [Werle et al., 2006], cystatin SN is upregulated in gastric cancer [Choi et

al., 2009], cystatin C [Sokol and Schiemann, 2004], cystatin M [Zhang et al., 2004],

and cystatin F [Utsunomiya et al., 2002] were found to be expressed in epithelial and


38

mesenchymal tumour cells. Cystatin M is often hailed as tumour suppressor. However

in certain cases cystatin levels appear to coincide with increased tumourigenicity

[Vigneswaran et al., 2003]. Stefin A and cystatin C overexpression has been shown to

inhibit cancer cell invasion and metastasis [Li et al., 2005; Kopitz et al., 2005].

Cathepsin B secretion may be important in penetration of the extra cellular

matrix during metastasis, and cystatin C may be involved in regulating this process

[Corticchiato et al., 1992; Dickinson, 2002]. The up regulation of cystatin F and down

regulation of cystatin M associated with cancer is also indicative of a functional

association. A correlation between high serum levels of cystatin C and higher risk of

death in colorectal cancer patients has been found [Kos et al., 2000].

Cysteine proteinases such as cathepsins B and L have been found to be

elevated in cancer. Elevated activities could be due to impaired regulation by the

endogenous low molecular mass cysteine proteinase inhibitors (cystatins). It was

found that the extract of cystatins from sarcoma was less effective against papain and

cathepsin B than was extract from liver. Inhibitory property of two members of the

cystatin superfamily (stefin A and stefin B) was determined. It was found that stefin B

from liver and sarcoma exhibited comparable inhibition of papain and cathepsin B. In

contrast, stefin A from sarcoma exhibited a reduced ability to inhibit papain, human

liver cathepsins B, H, L, human and murine tumor cathepsin. The Ki for inhibiton of

liver cathepsin B by sarcoma stefin A was 10-fold higher than that for inhibition of

liver cathepsin B by liver stefin A, reflecting a reduction in the rate constant for

association and an increase in the rate constant for dissociation [Lah et al., 1989].

Cystatin in brain diseases

Cysteine proteinases are implicated in various pathologies of the brain.

Alzheimer’s disease and many other neurodegenerative disorders are associated with

the accumulation of abnormal protein assemblies in the central nervous system.

Hereditary cystatin C amyloid angiopathy (HCCAA) and Progressive myoclonus

epilepsy (EPM1) are the genetic disorders caused due to mutation in cystatin C and

cystatin B genes, respectively [Cox, 2009].


39

Cystatins and amyloid fibrillation

Progressive mycolonus epilepsy of type 1 (EPM1) Human stefin B a

cysteine proteinase inhibitor is an intracellular protein expressed in many types of

cells, located in the cytoplasm and nucleus. It has been found as part of a multi protein

complex specific to the central nervous system [Giaimo et al., 2002]. The main

pathology for this proteinase inhibitor is its role in monogenic epilepsy, a progressive

mycolonus epilepsy of type 1 (EPM1), termed as Unverricht-Lundborg disease. There

are results that provide evidence that mutations in the gene encoding cystatin B are

responsible for the primary defect in patients with a progressive mycolonus epilepsy

of type 1 (EPM 1) [Len et al., 1996].

Cerebral amyloid angiopathy

Hereditary cystatin C amyloid angiopathy (HCCAA) is a rare, fatal

amyloid disease in young people in Iceland caused by a mutation in cystatin C, which

is an inhibitor of several cysteine proteinases such as cathepsins S, B and K. Mutated

cystatin C forms amyloid, predominantly in brain arteries and arterioles, but also to a

lesser degree in tissues outside the central nervous system such as skin, lymph nodes,

testis, spleen, submandibular salivary glands and adrenal cortex. The amyloid

deposition in the vessel walls causes thickening of the walls leading to occlusion or

rupture and resulting in brain haemorrhage [Palsdottir et al., 2006].

Immuno modulation

Cystatins have emerged as effector molecules of immunomodulation

[Zavasnik-Bergant, 2008]. They can stimulate nitric oxide release from macrophages

[Verdot et al., 1999]; modulate respiratory burst and phagocytosis in neutrophils

[Leung-Tack et al., 1990]; and modulate interleukin, cytokine production in T-cells

and fibroblasts [Schierack et al., 2003; Kato et al., 2002; 2004]. Most of these

functions operate via putative cell surface cystatin-binding molecules or membrane

domains [Kato et al., 2002].

Cystatin C has been shown to be a T-cell growth factor TGFβ receptor

antagonist and TGFβ signalling pathway blocker [Sokol and Schiemann, 2004; Sokol


40

et al., 2005]. Type 2 cystatins are also known to increase interleukin-6 (IL-6)

expression in fibroblasts and splenocytes [Kato et al., 2000].Cystatin C is a potent,

reversible inhibitor in vitro of the human lysosomal CPs e.g., cathepsin S (Ki = 8

pM), cathepsin L (Ki = 8 pM) and cathepsin H (Ki = 220 pM). These proteinases are

located all along the endocytic pathway of dendritic cell and are involved in the

controlled proteolysis associated with the degradation of antigenic peptides [Pluger et

al., 2002]. Cystatin F targeting of cathepsin C is known to regulate diverse immune

cell effector functions [Hamilton et al., 2008].

Cystatins and injury

Cystatins C appears to be up-regulated in response to injury in the brain.

Cystatin C protein was detected by immunohistochemistry in few of the hippocampal

pyramidal cells of the normal rat brain, but was present in these cells 3 days after

experimental ischemia [Palm et al., 1995]. It was localized to morphologically

degenerative neurons, and absent from morphologically viable neurons. Individuals

affected of Alzheimers disease shows strong localization of cystatin C protein in the

pyramidal neurons regions of the brain found the most susceptible to cell death in this

disease unlike the unaffected individuals [Deng et al., 2001]. The exact function of

cystatin C in the brain and its role in injury are presently unknown. Its appearance

would be consistent with a protective role, perhaps by blocking cysteine proteinase

activity in damaged cells to allow for recovery or by acting as a growth factor.

However, based on its association with damaged cells, it is conceivable that it may be

a mediator of injury.

Role of cathepsins and cystatins in patients with recurrent miscarriages

Cathepsins and their endogenous inhibitors cystatins were evaluated in tissue

and serum of patients with recurrent miscarriages. Deciduas and villi were surgically

collected from 22 patients and 12 healthy women. Immunohistochemistry was

performed with antibodies against cathepsins, stefin A, (cystatin A and cystatin B)

and cystatin C. The concentrations of cathepsins, stefins and cystatin C were

measured by enzyme-linked immunosorbent assay. Serum level of cystatin C in 85

Japenese women with recurrent miscarriage was also measured. Staining of cathepsin

B, D, H, L, stefin B and cystatin C was observed in the cytoplasm of epithelial cells in


41

decidua. Stefin A was found to be expressed on the surface of the trophoblast. The

concentration of cathepsin B and H in patients decidua was found to be significantly

higher than in control individuals. The serum level of cystatin C was found to be

significantly lower in patients than in control individuals. These findings suggest that

the regulation of the cathepsin cystatin system may play an important role in patients

with recurrent miscarriages [Tamao et al., 2005]. A study was carried out by Despina

et al. [2007] to investigate circulating levels of cystatin C (an endogenous marker of

renal function) in mothers, foetuses and neonates from intrauterine growth restricted

(IUGR; characterized by impaired nephrogenisis) and appropriate for gestational age

(AGA) pregnancies. Cystatin C levels did not correlate with gestational age and did

not differ between males and females. Fetal cystatin C serum levels are lower in the

IUGR group, significantly decrease after birth and do not correlate with maternal

levels in both groups. However cystatin C levels positively correlate with respective

creatinine and urea levels in the perinatal period.

Cystatin C in clinical diagnostics

Cystatin C was the first protein to be used in clinical diagnostics. Levels of

cystatin C in various body fluids is used as a barometer of disease [Shah and Bano,

2008]. Recent studies indicate that serum cystatin C is a better marker of glomerular

filtration rate and is a stronger predictor of cardiovascular disease and mortality than

serum creatinine [Fried, 2009]. Cystatin C levels can also be used to reflect the

characteristics of peritoneal membrane in dialysis patients [Al-Wakeel et al., 2009].

Korolenko et al. [2008] recently found that serum cystatin C concentration can be

used as one of the prognostic criteria in patients with several kinds of hemoblastoses.

IL-6 levels along with that of cystatin C may be regarded as markers of increased

osteoblastic activity associated to bisphosphate treatments in prostrate cancer patients

with bone metastases [Tumminello et al., 2009]. Yang et al., [2009] found that

cystatin C levels decrease significantly in cerebrospinal fluids of patients with

Guillain-Barre syndrome and may be involved in its pathophysiology. Cystatin B was

found to be specifically over expressed in most hepatocellular carcinomas and alone

or in combination with α-fetoprotein may be a useful marker for diagnosis of the

diseases [Lee et al., 2008].


42

Cystatins as antimicrobial and antiviral agents

Horse-shoe crab hemocyte cystatin has antimicrobial activity against Gram

negative bacteria, with IC50s against S. typhimurium, E. coli and K. pneumoniae in

the 80-100 µg/ml range [Agarwala et al., 1996]. Both chicken and human cystatins

were found to inhibit the growth of P. gingivalis with an IC50 of 1.1 and 1.2 fM,

respectively [Blank et al., 1996]. Cystatin C is also an effective inhibitor of

replication of coronavirus [Collins and Grubb, 1998]. Profound alterations in levels of

cystatin A was found in dendritic cells of few AIDS patients [Alavaikko et al., 1985].

Sialostatin L displays anti-inflammatory role and inhibits proliferation of cytotoxic T-

lymphocytes [Kotsyfakis et al., 2006].

1.19 CYSTATINS IN VARIOUS OTHER DISEASES

Cathepsins are involved in the degradation of extracellular matrix, facilitating

the growth, invasion and metastasis of tumour cells and also in tumour angiogenesis

[Gocheva and Joyce, 2007; Mohamed and Sloane, 2006; Turk et al., 2004]. Stefin A

and cystatin C overexpression has been shown to inhibit cancer cell invasion and

metastasis [Kopitz et al., 2005; Li et al., 2005]. Cystatins may also inhibit cell

migration by interfering with cell signalling pathways, by direct cathepsin and calpain

inhibition [Cox, 2009; Kassis et al., 2002]. Lower expression of stefin A may play a

key role in tumour invasion, whereas stefin B concentration increases above normal

level in the macrophages of lung cancer patients [Lah et al., 1990].

Cystatins have also been implicated in other inflammatory reactions in the

skin [Hayashi 1975]. Alavaikko et al [1985] reported that one or two cases of AIDS

showed profound alterations of dendritic reticulum cells revealed by localization of

cystatin A. Spanier and Bird [1982] indicated that the levels of cystatin decreases in

the guinea pig with a nutritional muscular dystrophy induced by vitamin E deficiency.

Cystatin A level increases in patients with cardiovascular diseases, sarcoidosis and

hepatic cirrhosis [Hopsu-Havu et al., 1983]. The concentration of cystatin B increases

in patients suffering from inflammatory diseases and kidney failure [Hapsu-Havu et

al., 1982]. There are many other diseases with decreased cystatin levels, such as

inflammatory diseases, osteoporosis, arthritis, and diabetes as well as a number of

other neurodegenerative diseases [Turk et al., 2008].


43

1.20 MUSCLE

Muscle derived from latin word “musculus” is a contractile tissue of animals

and is derived from the mesodermal layer of embryonic germ cells. Muscle cells

contain contractile filaments that move past each other and change the size of the cell.

Muscle plays a central role in whole body protein metabolism by serving as a

principal reservoir for amino acids to maintain protein synthesis in vital tissues and

organs such as skin, brain, heart and liver.

1.21 FUNCTIONAL ARCHITECTURE OF SKELETAL MUSCLE

Skeletal muscle cells may have the most highly ordered internal structure of

any cell in the body. A striated muscle consists of many parallel striated muscle fibres

united together in bundles termed fasciculi by connective tissue. Each muscle fibre is

an externally long, and fairly thick, multinucleate cell. The fibres are surrounded by

an electrically excitable, specilaized cell membrane, know as sarcolemma. The

numerous nuclei are arranged about the periphery of the fibre.

When an individual muscle fibre is examined microscopically, it is found to

consist of a bundle of many myofibrils that run parallel to one another throughout the

length of the fibre [Berry, 2002]. In a cross-section of fibre, the cut ends of the fibrils

look like dots. The myofibrils are embedded in an specialized intracellular fluid

termed the sarcolpasm. The sarcoplasm present in muscle fibres contain mitochondria

which produce large amounts of adenosine triphosphate (ATP) which is required for

muscle contraction.

Examination of stained muscle fibres in the electron shows the banding pattern

to be the result of the partial overlap of two distinct types of filaments, thin filaments

and thick filaments [Fig. 10]. Each sarcomere extends from one Z line and contains

several dark bands and light zones. A sarcomere has a pair of lightly staining I bands

located at its outer edges, a more densely staining A band located between the outer I

bands and a lightly staining H zone located in the centre of the A band. A densely

staining M line lies in the centre of the H zone. The I band contains only thin

filaments and a part of the A band on either side of the H zone, which represents the

region of overlap and contains both types of filaments.


44

The thin filaments contain the proteins actin, tropomyosin and troponin. The

thick and thin filaments interact by cross-bridges which emerge at regular intervals

from the thick filaments. Cross bridges are the sites at which the contractile force is

generated [Lakik, 1971].

1.22 PROTEIN FOLDING AND UNFOLDING STUDIES

The second chapter of the thesis is related to the cystatin folding and unfolding

studies in which investigation were carried out to study the behaviour of GMC upon

treatment with denaturants (urea and GdnHCl) to gain an insight into the folding

pathways adopted by GMC. Proteins are synthesized as a linear chain of amino acids

which in order to become biologically active must fold and adopt one out of an

enormous number of possible conformations. This conformation referred to as native

state, exists in solution as a very compact and highly ordered structure. Under

physiological conditions the native (folded) and the denatured (unfolded) states of a

protein are in equilibrium and the free energy change, ΔG, for the equilibrium

reaction is referred to as the conformational stability of a protein.

Native (N) Denatured (D)

The determinants of native state stability in aqueous solutions are the amino

acids sequence of protein as well as the variable conditions of pH, temperature, and

the concentration of salts and ligands [Alber, 1989; Pace, 1990]. Although the native

conformation is essential for activity, the conformational stability is remarkably low.

The native state of most naturally occurring proteins is only about 5-15 kCal/mol

more stable than its unfolded conformations [Pace, 1975]. Unfolding of small

compact proteins generally follow a simple two state transition, in such cases the

probability of all the intermediates between native and denatured states are very low

[Privalove, 1979; Privalove and Khechinashvili, 1974]. However it is now accepted

that under different conditions different states have been observed that do not appear

to be native or completely unfolded [Tanford 1968]. Intermediate states have been

found for several proteins like bovine and human carbonic anhydrase [Jagannadham

and Balasubramaniam, 1985; Wong and Tanford, 1973], β-lactamase [Michinson and

Pain, 1985; Robson and Pain, 1976] and bovine growth hormone [Brems et al., 1985].


45

Figure 10: Diagrammatic representation of the parts of muscle.

Muscle belly splits into various component parts:

a) Fasciculi

b) Muscle fibres

c) Myofibrils

d) Myofilaments

(a)

(b)

(c)

(d)


46

It is well known that proteins unfold under the action of denaturants like

GdnHCl and urea. Analysis of solvent denaturation curves using these reagents can

provide a measure of the conformational stability of a protein [Yao and Bolen, 1995;

Pace 1986]. Despite the wide spread work in this front, description of the interaction

of GdnHCl and urea with proteins is not complete owing to the complex structure of

folded proteins. There are various reports available which have shown different

effects of GdnHCl and urea on protein unfolding like mushroom tyrosinase which

shows different behaviour towards GdnHCl, urea and SDS in terms of different

transition processes for these denaturants [Park et al., 2003]. Behaviour of human

placental and goat lung cystatins towards these denaturants was also found to be

different in terms of midpoint of transition and presence of intermediate states [Rashid

et al., 2005; Khan and Bano 2009].

Though the partially folded forms of various proteins have been characterized,

it has not yet been possible to generalize the concept of ‘intermediate states’ as

universal equilibrium protein folding intermediates. The molten globule (MG) is an

equilibrium state of many proteins under mild denaturing conditions and a universal

kinetic intermediate of protein folding [Ptitsyn, 1987 and Kuwajima, l989]. Its

properties are intermediate between those of native (N) and unfolded (U) protein

molecules: it is compact [Dolgikh et al., 1981 and Gast et al., 1986] and has a number

of important features of the native secondary structure and of the native overall

architecture [Ptitsyn, 1987] but has only traces of the detailed native tertiary structure.

These properties mean that the molten globule is structually quite different both from

the native protein and from the unfolded chain. Urea- andGdnCl-induced denaturation

of proteins leads to their more or less complete unfolding. This unfolding can occur

either in one step (an N→U transition, [Tanford 1968 and Privalov, 1979] or in two

separate steps (N→MG and MG→U transitions, see [Christiensen and Pain, 1991]

which take place at different concentrations of denaturing agents, although they can

partially overlap. This provides a good opportunity to study the N→MG and MG→U

transitions separately.

Increasing inclination in protein folding/unfolding is due to the recognition

that failure of cellular protein folding mechanisms is associated with a variety of

important human disorders ranging from cystic fibrosis to Alzheimer’s disease.


47

There is a growing body of evidence indicating a critical role for partially folded

protein conformers in the process of conversion of normal cellular proteins into

disease causing, protease-resistant protein aggregates of various morphologies [Kelly,

1998; Dobson, 2004].

Thus, in this study a comprehensive analysis involving the various

conformational changes in goat muscle cystatin (GMC) on interaction with GdnHCl

and urea have been undertaken to explore the hierarchy of protein folding.

1.23 INTERACTION OF PROTEIN WITH DRUGS

Third chapter of the thesis deals with drug protein interaction. Keeping in

view the fact that (MTX) acts as an anti-arthiritic drug and DXN is used for the

treatment of muscular dystrophy their interaction with muscle cystatin aroused great

interest. The binding and interaction of drugs with plasma and tissue proteins strongly

affects their distribution, metabolism as well as pharmacodynamics and toxic

properties. Accumulation of drug molecules at certain sites in the body causing a

localized high concentration, adverse drug reactions [Wen and Ye, 1993] and ligand

induced protein structure conformational changes [Takeda et al., 1988] are major

problems complicating drug medical therapy. Therefore, studies analyzing the binding

mechanism between proteins and drugs and the structure of resulting complexes are of

particular interest. These studies enable to elucidate how ligand affinity is regulated

and how the protein conformation is altered upon complexation. Investigating the

properties of drug-protein complex may provide useful information that determines

the therapeutic effectiveness of drugs.

Methotrexate (4-amino-10-methylfolic acid) chemical structure shown below,

also known as amethopterin is a chemotherapeutic drug used to treat leukemia,

lymphomas, and osteosarcoma. It is also used in the treatment of AIDS and

rheumatoid arthritis. Methotrexate is a folic acid antagonist and competitively inhibits

folic acid reductase which alters folic acid into tetrahydrofolic acid, which in turn

required for the synthesis of coenzyme used in the synthesis of DNA.Though,

methotrexate has been widely used as anticarcinogen and in the treatment of arthritis

due to its economical nature (also known as poor man drug), it causes many side


48

effect which commonly include abnormal liver function, Immunosuppression,

neurotoxicity and lung damage [Hughes et al., 1987].

Chemical structure of methotrexate

Dexamethasone (DXN) is a potent synthetic member of the glucocortocoid

class of steroid harmone, chemical structure shown here It acts as an anti-

inflammatory and immunosuppressant [Naik, 2010]. It is synthesized corticosteroid

compound that has been widely used in clinical treatment for muscular atropy,

Addison’s disease, systemic lupus erythematosus and other auto-immune diseases.

Chemical structure of dexamethasone


49

1.24 EFFECT AND FUNCTION OF ALCOHOLS AND SALTS ON

PROTEIN STRUCTURE

The aim of the present investigation in fourth chapter was to study the acid

induced unfolding of cystatin at low pH. Further the influence of alcohols (methanol,

sorbitol and PEG-200) and salts (Na2SO4, KCl and KBr) was checked on the low pH

induced state of muscle cystatin to gain insight into its conformational stability.

Proteins carry out the most considerable tasks in living organisms so stabilizing the

protein structure is an important issue in biological systems. The unfolding/refolding

of proteins by chemical denaturants under in vitro conditions has received greater

attention as various partially folded intermediates have been found to accumulate

during equilibrium unfolding of a number of proteins [Uversky et al., 1998: Fink et al

1997; Muzammil et al., 2000].

There has been a considerable interest in characterizing these partially folded

states for gaining an insight into the possible determinants of the protein folds and the

mechanism of protein folding [Mizuguchi, 2000; Uversky, 1999]. Characterization of

partially folded intermediates is relatively complex in multi-domain proteins where

each domain is capable to unfold/refold independently and the association of different

domains in the whole protein through various short range and long range interactions

may affect the overall folding topology of the protein [Mizuguchi, 2000; Dockal,

2000]. It has been demonstrated that residual structural preferences, ranging from

local clusters of side chains to highly ordered sub-domains persist in denatured states

of proteins [Dobson, 1991; Redfield et al., 1994; Ptitsyn, 1995].

pH is known to influence the stability of a protein by altering the net charge on

the protein. Many proteins denature at extreme pH values because of the presence of

destabilizing repulsive interactions between charges in the native protein [Creighton,

1993; Tan et al., 1995]. The exact behaviour of a given protein at low or high pH is a

complex interplay between a variety of stabilizing and destabilizing forces, some of

which are sensitive to the environment. Acid denaturation results in a denatured state

that is often less unfolded than the completely unfolded form obtained in high

concentration of urea and GdnHCl [Rashid et al., 2005]. In acid denaturation the

major driving force for unfolding is intramolecular charge repulsion which may fail to


50

overcome interactions that favour folding such as hydrophobic forces, disulphide

bonds, salt bridges and metal ion protein interactions. The mechanism by which,

proteins fold from a structureless, denatured state to their unique biologically active

states is an intricate process. However, recent advances in biophysical techniques

[Nolting et al., 1995; Plaxcon and Dobson, 1996] both thermodynamic and kinetic

studies, have shown the presence of stable intermediate conformational states in a

number of proteins [Ptitsyn, 1994; Radford and Dobson, 1995] which helped in the

understanding of protein folding phenomena.

Proteins are stabilized generally by a combination of hydrogen bonding,

electrostatic interactions and hydrophobic interactions. Various studies have been

made for in improving the protein stability and it has been established that protein

stability is the result of balance between intramolecular interaction of protein

functional groups and their interaction with solvent environment [Yancey and

Somero, 1980; Timasheff 1993; Zou et al., 2002]. An extensively studied co-solvent

that modifies the protein conformation is alcohol. Studies on alcohol effects provide

insights into biologically important events because the alcohol solution mimics the

environment of biomembrane [Bychkova et al., 1996] and modifies folding pathways

of proteins [Chiti et al., 1999; Lu et al., 1997].

Alcohols exert several distinct effects on proteins and have been commonly

used to study partially folded states in proteins, thus creating better understanding of

the protein folding mechanism [Shiraki et al., 1995; Schonbrunner et al., 1996].

Binary mixtures of water with alcohols like methanol, ethanol or 2, 2, 2-

trifluoroethanol (TFE) denature the tertiary and quaternary structures of proteins

while enhancing their helicity [Radford and Dobson, 1995]. Alcohols promote local

polar interactions and hydrogen bonds in proteins, at the same time weakening non-

local hydrophobic interactions [Dill et al., 1995; Liu and Bolen, 1995]. The effect of

various polyols such as ethylene glycol, glycerol, erythritol, xylitol, sorbitol and

inositol have been studied on the structure of acid unfolded horse cytochrome C at pH

2.0 in order to address the contribution of hydrophobic interactions to the stability of

molten globule state of proteins [Kamiyana et al.,1999].

Physical and chemical properties of proteins can also be altered by polymer

interaction [Raqi, 2005]. The PEG polymers are a series of non-ionic polyhydroxyl


51

compounds that are miscible with water and display colligative properties in solution.

Different molecular weight PEG’s exhibit different solubilities, surface tension,

viscosities, freezing points and melting points. Salts have been known to affect the

physicochemical properties of proteins like their solubility [Carbonnaux, 1995]

stability [Von Hipple and Schleich, 1969] and pKa [Abe et al., 1995; Tan et al., 1995]

to a great extent. Salts can induce conformational transitions in alkaline or acidic

solutions from a largely unfolded state to an intermediate conformational state [Bai et

al., 1998]. Salts effect mainly the electrostatic and hydrophobic interactions in the

protein molecules. In the presence of salts, a conformational transition at acidic or

alkaline pH from a largely unfolded state to an intermediate conformational state has

been reported for several small globular proteins [Fink et al., 1994; Goto et al., 1990;

Babu and Bhakuni, 1997]. The main forces unfolding the protein under extreme pH

conditions are the repulsive forces between charged groups on the protein molecule.

Under these conditions the presence of higher salt concentration will have two effects

on the protein conformation. The Debye-Huckel screening effect, ie the counter ions

presence around the charged groups will weaken the repulsion thus permitting the

other forces favoring folding to become relatively strengthened and resulting in the

folding of the protein. In addition of this non-specific effect of salts, specific effects

of salts on protein folding will come into play. The stabilizing and destabilizing

effects of salts on proteins arise either by effects on water surface, and hence

hydrophobic interactions or by specific interactions with charged groups.

It is well established that the effect of anions in stabilizing protein structure

follows the Hofmeinster series [Von Hipple and Wong, 1965]. The representative

series is sulphate> phosphate> fluoride> chloride> bromide> iodide> perchlorate>

thiocyanate. In this series the ions to the left of chloride stabilize the native structure

of the protein, whereas the anions to the right of chloride destabilize the native

structure of proteins. Arakawa and Timaseff [1982] have shown that preferential

hydration of protein occurs in the presence of salts such as NaCl, sodium acetate and

sodium sulphate and the resulting unfavourable free.

Energy of the unfolded state is related to the stabilization effects of these salts

on the proteins. Salt dependent conformational transitions at alkaline or acidic pH

may be a general property of proteins, however, the concentration of salt required to


52

bring about the transition will depend on the particular protein being studied and the

conditions employed. Therefore, the effects of polyols and salts have been studied on

refolding of acid denatured muscle cystatin.

1.25 REACTIVE SPECIES MEDIATED PROTEIN DAMAGE

In the fifth chapter of thesis an attempt has been done to investigate the

damage of cystatin by reactive oxygen and reactive nitrogen species. Muscle tissue is

highly exposed to free radicals owing to oxidative stress as a result of sternous

exercise. Thus an investigation was carried out in which we exposed GMC to free

radicals and also explored the anti-oxidant potential of green tea flavanoids EC and

EGCG. Radical mediated damage to proteins, initiated by electron leakage, metal-ion-

dependent reactions and auto-oxidation of lipids and sugars results in production of

protein hydroperoxides and aggregation or fragmentation of proteins. Damaged

proteins are often functionally inactive and their unfolding is associated with

enhanced susceptibility to proteinases.

Reactive species

Free radicals such as reactive oxygen (ROS) and reactive nitrogen species

(RNS) are well recognised for playing a dual role as both deleterious and beneficial

species [Valko et al., 2007]. Free radicals are also involved in ATP generation,

apoptosis of effete or defective cells, production of prostaglandins and leukotrienes

[Devasagayam et al., 2004].

Nature has endowed cells with protective antioxidant mechanisms to

neutralize and eliminate the harmful reactive species. However, oxidative stress can

occur when there is a disturbance in the pro-oxidant/antioxidant systems in favour of

the former [Sies, 1985]. Overproduction of RNS is termed as nitrosative stress

[Ridnour et al., 2004]. The uncontrolled oxidative stress initiates a series of harmful

biochemical events associated with diverse pathological processes [Juranek and

Bezek, 2005].

Reactive species are small molecules with an oxygen or nitrogen atom in their

structure [Halliwell and Gutteridge, 2007]. These ROS and RNS can be free radicals


53

with an unpaired electron [e.g. •NO (nitric oxide radical), O2•‾ (superoxide radical)

and OH• (hydroxyl radical)] or non-radicals (e.g. H2O2). They can be anions [e.g. O2‾

(superoxide) ONOO‾ (peroxynitrite)] or non-ions (e.g. H2O2, •NO, OH•).

1.26 EFFECT OF NITRIC OXIDE (NO) ON GMC AND ITS

RECLAMATION BY EPIGALLOCATECHIN GALLATE

(EGCG) AND EPICATECHIN (EC)

Niric oxide (NO), an atmospheric gas and free radical is also an important

biological mediator in animals and humans. It is synthesized from semi-essential

amino acid L-arginine by NO synthases (NOS), a family of enzymes with four major

types from different origins: endothelial, neuronal, inducible and mitochondrial. They

can be found in almost all tissues and they can coexist in the same tissue. NO is

thermodynamically unstable molecule and tends to react with other bio molecules

especially proteins resulting in their oxidation, nitration and nitrosylation, with the

concomitant effects on many cellular mechanisms. Nitric oxide is an omnipresent

intercellular messenger in all vertebrates, modulating blood flow [Huang et al., 1994],

thrombosis [Pacher et al., 2007] and neural activity [Bredt and Synder, 1994]. The

production of nitric oxide is also important for non-specific host defence, helping to

kill tumours and intracellular pathogens. However, under pathophysiological

conditions NO has damaging effects. It is found to be involved in neural disorders

involving oxidative stress such as Parkinson’s disease [Moreau and Huber, 1999],

Alzheimer’s disease [Tran et al., 2003], Ischemia [Thiyagarajan et al., 2004], multiple

sclerosis [Kahl et al., 2003], and pulmonary hypertension [Giaid and Saleh, 1995].

Excessive production of NO (a known mediator of inflammation) is correlated with

nitostative stress in tissues. Although NO˙ is a free radical, it has selective reactivity

and reacts predominantly with other paramagnetic species, including ferrous or ferric

iron in heme proteins or iron-sulphur centres, and other radical species such as

molecular oxygen (O2), superoxide anion (˙O2-) and lipid or protein radicals

[Kharitonov et al., 1994; Rubbo et al., 1966]. Reaction of NO with ˙O2- at a near

diffusion limited rate yield peroxynitrite (ONOO-), a powerful oxidizing species, to

which many of the cytotoxic properties of NO˙ have been attributed [Beckman and

Koppenol, 1996]. In healthy human subjects, (NO2-) nitrite can be detected at levels of

approximately 15µM in respiratory tract living fluids [Pryor and Squadrito, 1995]


54

which is raised to approximately 36µM in patients with interstital pneumonia [Gaston

et al., 1993].

The redox situation within the tissues is a dynamic balance (homeostasis)

between the production of reactive oxygen species and anti-oxidant system capacity

and this determines the level of oxidative damage. The skeletal muscle is the largest

consumer of oxygen in the body. The oxidative capacity of the skeletal muscle

reflects the capacity of a working muscle to synthesize adenosine triphosphate (ATP)

through aerobic metabolic pathways. ROS-mediated damage is the result of an

increase in electron flux and the corresponding leakage from the mitochondrial

respiratory chain. The skeletal muscles (as well as the central nervous system) are

particularly vulnerable to “oxidative stress” because they are tissue made up of post

mitotic cells that use a large amount of oxygen, thus causing cumulative oxidative

damage to the cell structures over time. Furthermore, it has been suggested that the

dynamic ability to change the mitochondrial oxidative capacity, typical of this tissue,

in response to variations in energy demands makes the tissue particularly prone to

ROS-mediated damage.The antioxidant reserve capacity in most tissues is not

marginal, but during aging the production of ROS presents a challenge to the

antioxidant systems because it could exceed their capacity [Fig. 11].

Knowledge about prevention of protein oxidation is cursory. Certain

approaches attenuate the secondary radicals and confer cytoprotection [e.g.

mesalamine against ONOO‾] but fail to affect the radical sinks [•NO levels remain

unaffected] [Sandoval et al., 1997].

Antioxidant agents of natural origin like polyphenols and flavonoids etc. have

attracted special interest lately, because of their high efficacy and multifaceted health

benefits [Ullah and Khan, 2008]. Various researches have been undertaken to evaluate

the potential benefits of complimentary and alternatives medicine (botanicals and

flavonoids herbs) to oppose the toxicity associated with reactive species generated

during various pathogenic states.


55

Figure 11: Diagrammatic representation of the cell damage by free

radicals and protection by antioxidants.


56

Green tea (Camellia sinenis) is one of the most commonly consumed

breverages in the world and is a rich source of polyphenols, including catechins,

theaflavins and thearubigins. Tea polyphenols act as antioxidants in vitro by

scavenging reactive oxygen and nitrogen species and chelating redox- active

transition metal ions.The principal catechins found in tea are epigallocatechin gallate

(EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and epicatechin (EC).

EGCG (chemical structure shown below) is the most abundant catechin in tea

[Graham, 1992]. It constitutes upto 63% of total catechins [Manning and Roberts,

2003]. EGCG has been shown to be 25 to 100 times more potent than vitamin C and E

in terms of antioxidant activity [Doss et al., 2005]. A cup of green tea typically

provides 60 to 125mg catechins including EGCG [Cooper et al., 2005].

Chemical structures of epigallocatechin gallate (EGCG) and

epicatechin (EC)

The anti-rheumatic potential of EGCG has been proved using in-vitro

experimentation. EGCG has been found to have cartilage preserving and

chondroprotective action [Ahmed, 2010]. Epicatechin is also a major polyphenolic

component of green tea and is reported to have insulin mimetic action with protective

effects on erythrocytes in a manner similar to insulin.


57

Epicatechin cause blood vessel dilation by regulating nitric oxide, which is

released by blood vessel endothelium to signal surrounding muscle to relax.

Epicatechin has been reported to have anti-oxidant capacity [Lee et al.,2003].

Epicatechin also offers anti-oxidant protection against lipid peroxidation and inhibits

platelet aggregation. High quantities of epicatechin can also be found in coca and

grapes. Dreosti [2000] reported that 60% of the total phenolic compounds in raw coca

beans is flavanol monomers (Epicatechin and Catechin). Epicatechin has been

reported to be a potential candidate to scavenge free radicals in our body and food

systems [Sanbogi et al., 1998]. In vitro studies demonstrated that these compounds

have several biological activities such as the ability to scavenge superoxide anion and

hydroxyl radicals, to reduce lipid peroxyl radicals and inhibit peroxidation [Salah et

al., 1995].

1.27 SCOPE OF THE THESIS

In consideration of the fact that ubiquitously proteinase inhibitors cystatins are

involved in myriad of physiological processes and implicated in a number of diseased

conditions, it was thought worthwhile to purify cysteine proetinase inhibitors from an

important source, muscle, with a view to gain insight into the regulation of cysteine

proteinases in this organ. In order to have an in-depth knowledge about the

complexities and the physio-chemical characteristics of cysteine proteinase inhibitor.

Isolation and purification of cysteine proteinase inhibitors from goat muscle

attained through gel filtration chromatography yielded a single specific peak,

designated as goat muscle cystatin (GMC). Its detailed biochemical characterization

was undertaken, which included elucidation of its molecular weight, pH, temperature

and thermal stability. Its interaction with model cysteine proteinase, papain was

worked out to determine its kinetic properties. To assess the conformational stability

of the isolated inhibitor, equilibrium denaturation studies were conducted with

classical denaturants, guanidine hydrochloride and urea employing fluorescence and

CD spectroscopy.

Drug-protein interactions are determining factors in the therapeutic,

pharmacodynamic and toxicological drug properties. Experiments were conducted to


58

find out the effects of drugs MTX and DXN on structure and function of the purified

inhibitor employing UV and fluorescence spectroscopy.

GMC was found vulnerable to oxidative stress as depicted by activity and

fluorescence measurements. Green tea flavonoids, EC and EGCG offering

inexpensive and non-toxic modes of anti-free radical therapy were analyzed to

ascertain their efficacy and efficiency in preventing ROS induced damaged to the

isolated inhibitor. Studies on the effects of alcohols on muscle cystatin can provide

insights into the biologically important events because the alcohol solution mimics the

environment of biomembrane and modifies folding pathways. The refolding studies of

GMC induced by salts may prove useful in understanding of the pathway by which

the proteins attain their native conformation and various conditions that can be

employed to refold the denatured protein back to its native state.

The studies are worthy of future research with regard to the interaction of

muscle cystatin with muscle cathepsins and its role in muscle (patho) physiology.

Introduction - shodhganga.inflibnet.ac.inshodhganga.inflibnet.ac.in/bitstream/10603/13140/10/10_introduction.pdfAatif M. Ph.D Thesis Introduction 2 Cysteine proteinases (CPs) are the

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